The effect of central loops in miRNA:MRE duplexes on the efficiency of miRNA-mediated gene regulation - PubMed (original) (raw)
The effect of central loops in miRNA:MRE duplexes on the efficiency of miRNA-mediated gene regulation
Wenbin Ye et al. PLoS One. 2008.
Abstract
MicroRNAs (miRNAs) guide posttranscriptional repression of mRNAs. Hundreds of miRNAs have been identified but the target identification of mammalian mRNAs is still a difficult task due to a poor understanding of the interaction between miRNAs and the miRNA recognizing element (MRE). In recent research, the importance of the 5' end of the miRNA:MRE duplex has been emphasized and the effect of the tail region addressed, but the role of the central loop has largely remained unexplored. Here we examined the effect of the loop region in miRNA:MRE duplexes and found that the location of the central loop is one of the important factors affecting the efficiency of gene regulation mediated by miRNAs. It was further determined that the addition of a loop score combining both location and size as a new criterion for predicting MREs and their cognate miRNAs significantly decreased the false positive rates and increased the specificity of MRE prediction.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Validation of VEGF regulation by putative miRNAs.
The effect of putative VEGF-regulative miRNAs on VEGF expression was tested in hypoxia-induced CNE cells by transfecting the cells with siRNA duplexes homologous in sequence to the miRNAs in group 1 (A) and 2 (B). VEGF expression was determined by ELISA. MiRNAs in group 1 were selected using a strict criteria: free energy less than −24 kcal/mol, nt2–7 perfectly pairing with the 5′-end of miRNAs, and sequence conservation of target sites across five vertebrate species. MiRNAs in group 2 were chosen from miRNAs that only met relatively relaxed criteria: free energy between −24 kcal/mol and −15 kcal/mol, and an elastic seed window tolerating one G:U wobble in successive 6-mers. Repressive ratio = (1–ELISA value of miRNA/ELISA value of blank)×100%. The blank is the sample from cells without transfection, providing a control for protein expression in the absence of regulation. A random sequence (NC), miR-224, mutated miR-16 (miR-16-M), and miR-20a (miR-20a-M) that do not have MREs in the VEGF 3′-UTR were used as negative controls.
Figure 2. Luciferase activity assay.
COS-7 cells were co-transfected with the luciferase reporter vector, which contained either the VEGF 3′-UTR fragment nt31-216 (pRL-VEGF-Con1) or nt703-944 (pRL-VEGF-Con2), and an miRNA which has a putative binding site in either pRL-VEGF-Con1 (A) or pRL-VEGF-Con2 (B). Luciferase activity was measured to determine the effect of these miRNAs on luciferase translation. All miRNAs have only one predicted MRE in the corresponding report vector. Repressive ratio = (1–LA value of miRNA/LA value of blank)×100%. LA: luciferase activity. The blank is the sample from cells without transfection, providing a control for protein expression in the absence of regulation. A random sequence (NC), miR-29b, miR-150, miR-106b, and miR-134 that do not have MREs in the VEGF 3′-UTR were used as negative controls.
Figure 3. Correlation and linear regression analysis of central loops of miRNA:MRE duplexes.
A, Central loops (or bulges) of miRNA:MRE duplexes were divided into three categories: standard loop, type I decentered loop, and type II decentered loop. Standard loops start at between nt9 and nt11 of the miRNA:MRE duplex counting from the 5′ end of the miRNAs. The type I decentered loop starts before nt9, with the type II decentered loop opening after nt11. The loop score is designated according to the algorithm introduced in the Methods. B–D, Relationship between miRNA repression efficiency and the central loop score (B), tail score (C), and minimum free energy (D). Compared with the tail score and minimum free energy, the central loop score has a closer correlative relationship with miRNA repression efficiency (r = 0.646, p<0.001).
Figure 4. Mutation assays.
Site-directed mutagenesis of miR-17-5p (A) , miR-372 (B), pRL-VEGF-Con1 (D), and pRL-VEGF-Con2 (E) caused movement of the standard loops of miRNA:MRE duplexes, forming Type I and Type II decentered loops instead, while the free energy of miRNA:MREs was kept at similar levels. COS-7 cells were transfected with wild type or mutated miRNAs and different report vectors. The levels of luciferase activity decreased significantly due to the change in loop location (C and F).
Figure 5. Effect of loop location on miRNA functioning.
The insert fragments of the VEGF 3′-UTR in pRL-VEGF-Con1 and/or pRL-VEGF-Con2 were changed to fragments of the c-Met 3′-UTR or COX-2 3′-UTR to create pRL-CMET-Con3 and pRL-COX2-Con4. These changes allowed us to compare miRNA:MRE duplexes bearing standard central loops (A and D) in miRNA:MRE duplexes with the ones bearing Type I (B) and Type II (E) decentered loops. Luciferase activity assays indicated that changes to Type I and Type II decentered loops decreased the repressive effect of miRNAs significantly (C and F).
References
- Seggerson K, Tang L, Moss EG. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev Biol. 2002;243:215–225. - PubMed
- Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell. 2002;9:1327–1333. - PubMed
- Yekta S, Shih IH, Bartel DP. MicroRNA-directed cleavage of HOXB8 mRNA. Science. 2004;304:594–596. - PubMed
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